The main work carried out was the production of HIV and HIV-related proteins by recombinant DNA methods for high-resolution structural analyses. Proteins were selected which are important for the life cycle of the virus and for its structural integrity and, thus, represent potential targets for rational structure-based drug design. Collaborating groups (indicated in parentheses) performed the actual structure determinations. The determination of the 3-D structure of proteins by X-ray diffraction or multidimensional NMR required the production of large quantities of highly purified and physically homogeneous protein. The determination of protein structures by NMR required protein biosynthetically labeled with combinations of the stable isotopes: H-2, C-13 and N-15. (1) We have continued to initiate and support the structural analyses by NMR of HIV Nef, a 17 kDa protein essential for the pathogenic properties of the virus (A. Bax; Grzesiek ). The high-resolution structure determination was completed. We have also embarked on a more detailed analysis of HIV Nef in complex with SH3 and SH2 domains derived from the hck and lck Scr tyrosine kinases. It is hoped these studies will shed light on the biological consequences of these specific protein-protein interactions. (2) HIV Rev is an important regulatory factor required for HIV expression. This RNA binding protein has a strong tendency to self-associate into very high molecular weight polymers, which are unsuitable for analysis by NMR and X-ray crystallography. However, using spectroscopic methods and cryo-electron microscopy and image analysis, a low-resolution model has been derived. Using protein engineering and general biochemical approaches we are attempting to produce a low molecular form of the protein more suited for high-resolution structural studies (Watts; Steven). (3) In our continuing studies on the HIV protease we are currently analyzing novel mutants designed to stabilize it against proteolytic degradation. These mutants may facilitate structural-function studies by NMR based methods (London). (4) A structural study was initiated on the anti-HIV plant protein MAP30. This 30-kDa protein appears to inhibit HIV-1 integrase and other essential viral enzymes. Purification methods and biosynthetic labeling protocols were developed and the high-resolution NMR structure determination is well advanced (Torchia; S.Lee-Huang).